The overall goal of this proposal is to develop exosome-like nanoparticles as a delivery vehicle for targetable delivery of extracellular RNA (exRNA) primarily focusing on miRNAs which modulate gene expression and induce immune responses upon delivery in mouse tumor models. Development of exRNA-based therapeutics must overcome challenges including issues of delivery, potential off-target effects, safety, toxicity, the cost of large scale production, elimination of potential biohazards to the environmen and tissue specificity. Unlike the situation with artificially synthesized nanoparticles or liposomes, naturally derived nanoparticle-size exosomes are released from many different types of cells. miRNAs and RNAs can be encapsulated in naturally derived exosomes and the RNAs are stable, and a number of miRNAs can be naturally encapsulated simultaneously in these exosomes. Recently, data published by ours and other groups have demonstrated that mammalian exosomes can deliver poorly soluble agents, chemotherapeutic drugs, and siRNA in vivo. A limitation of this mode is the need for large-scale production of exosomes, and potential biosafety. Our preliminary data show that exosome-like nanoparticles can be isolated in large quantities from the tissue of edible plants, including grapes. We have demonstrated that exosome-like nanoparticles from grapes are composed of small RNAs, proteins, and lipids. An edible plant-derived nano-vector (EPNV) assembled from grape exosome-like derived lipids is capable of encapsulating siRNAs. Co-delivery of folic acid, with the chemotherapy drug paclitaxel by EPNVs results in an increased efficiency in tumor tissue targeting. Reducing the size of EPNVs by passing them through an appropriate pore size filter significantly enhances EPNV translocation to the brain and oral administration of the EPNVs results in their migration to the liver. Based on these preliminary data and the current literature, we propose to: (1) Determine whether an EPNV can deliver therapeutic miR17 via oral administration to target metastatic colon tumor in the liver, subsequently inducing NK cell activation to kill metastatic tumor cells; (2) Determine whether miR155 encapsulated in EPNV promotes differentiation of myeloid derived suppressor cells (MDSCs) into mature dendritic cells in mouse breast/brain tumor models; (3) Determine whether a therapeutic dose of miRNA17 or miRNA155 delivered by EPNV induces no side-effects in mouse tumor models; and (4) Determine whether EPNV can be produced in large scale amounts economically for use in clinical settings. Demonstration of using a EPNV-based delivery system loaded with a desired therapeutic extracellular RNA (exRNA) including microRNA to target to inflammatory cells and tumor cells in vivo would not only be a significant step forward in the treatment of cancer, but also provide a powerful tool for identifying the role and effects of each small RNA encapsulated in resident or circulating microvesicles on the recipient cells. We will accomplish this in our study by knock-out and knock-in strategies.